Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release.
Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release.
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分子伴侣 DnaK 的 D 和 E 螺旋对于 ATP 结合和底物释放的重要性。
DOI：
10.1021/bi034126v
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Witt,StephanN
中科院分区：
文献类型：
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作者：
Slepenkov,SergeyV;Patchen,Brandi;Peterson,KennethM;Witt,StephanN
分子伴侣DnaK的C-末端结构域是一个紧凑的盖状结构，由5个α-螺旋（αA−αE）（残基508−608）组成，随后是一个30个残基的无序柔性区域（609−638）。盖子封装了结合在底物结合结构域中的肽分子，而30个残基的无序区域的功能尚不清楚。通过依次删除柔性亚结构域和单个盖螺旋，我们推断出每个结构单元对创建长寿命DnaK−肽复合物的重要性。在这里，我们报告（i）αD螺旋是长寿命的DnaK−肽复合物所必需的。例如，ATP触发丙烯酰丹标记的p5肽（ap 5，a-CLLLSAPRR）从wtDnaK和DnaK 595（A-D）解离，koff分别等于7.6和8.9 s-1，而当D-螺旋缺失时，产生DnaK 578（A-C），koff跳至207 s-1。(ii)αB螺旋的存在影响ATP诱导的高亲和力至低亲和力构象变化的速率。例如，ATP在无盖变体DnaK 517（1/2A）中诱导这种构象变化，速率常数为442 s-1，而在添加回B-螺旋（残基518 - 554）后，ATP在DnaK 554（A-B）中诱导这种构象变化，速率常数为2.5 s-1。我们的解释是，这种大幅度下降的发生是因为DnaK 554的B-螺旋（A-B）结合在底物结合位点。(iii)缺失分析还显示，包含αE螺旋和柔性亚结构域的残基596−638影响ATP结合。我们的研究结果是一致的，这部分的盖子产生构象异质性，也许是通过结合到ATP酶结构域。
The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five α-helices (αA−αE) (residues 508−608) that is followed by a 30-residue disordered, flexible region (609−638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK−peptide complexes. Here we report that (i) the αD helix is essential for long-lived DnaK−peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A−D) withkoffequal to 7.6 and 8.9 s-1, respectively, whereas when the D-helix is deleted, creating DnaK578(A−C),koffjumps to 207 s-1. (ii) The presence of the αB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s-1, whereas, after adding back the B-helix (residues 518−554), ATP induces this conformational change in DnaK554(A−B) with a rate constant of 2.5 s-1. Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A−B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596−638, which comprise the αE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain.